Antigen-induced secretion of granules from cultured RBL-2H3 mast cells is dependent on calcium and protein kinase C. Secretion can also be induced by mast cell secretagogues such as compound 48/80, which act via Gi GTP-binding proteins, in phenotypically altered RBL-2H3 cells (by co-culture with fibroblasts or the kinase inhibitor, quercetin; J. Immunol. 160:5136,1998). The secretory response to all stimulants is enhanced by co-stimulation of cells with adenosine A3 receptor agonists and by prior treatment of cells with cholera toxin. (see previous reports in this series). We have examined the role of phospholipase(PL) D in secretion as this enzyme, rather than PLC, is primarily responsible for the generation diglycerides for the activation of protein kinase C and its activation is enhanced in cholera toxin-treated RBL-2H3 cells (J. Pharmacol. Exptl. Ther. 285:110, 1998). Adenosine analogs and compound 48/80 were found to elicit sustained activation of PLD and protein kinase C but only transient activation of PLC, calcium mobilization and secretion. The activation of PLD and secretion were enhanced in cholera toxin-treated cells and blocked in pertussis toxin-treated cells. These and other data indicated that these two stimulants and cholera toxin acted via Gi and Gs, respectively, and independently of cAMP. The possibility was tested that PLD may be activated by G-beta/gamma subunits released from Gi and Gs by overexpression of these subunits in the Semliki viral expression system. Such overexpression significantly enhanced basal and stimulated PLD activity in RBL-2H3 and BHK cells and synergised the effects of cholera toxin. The effects of the G-beta/gamma subunits were neutralized by co-expression of G-alpha-s. We believe that these results indicate for the first time that PLD is directly linked to receptors via trimeric GTP-binding proteins. Gene cloning revealed the presence of PLD1b and PLD2 in RBL-2H3 cells. Other studies with permeabilized cells (described in preliminary form last year; see Z01 HL 00937-14 LMI), have since confirmed the existence or two forms of PLD in RBL-2H3 cells. One form, presumed to be PLD1b, is dependent on the small G protein, ARF, for activation. The other is sensitive to cholera toxin, receptor-regulated, and most likely PLD2. Current work is focussed on mechanisms for the possible interaction of PLD2 with G beta-gamma subunits. An unexpected finding was that RBL-2H3 cells slowly secrete granules in response to very low concentrations of antigen. This stimulation was associated with slow induction of COX-2, an enzyme involved in the synthesis of prostaglandins (PG) particularly PGE2. Our interest here is that low levels of stimulation may lead to changes in mast cell responses in chronic allergic/inflammatory diseases.